In the macronuclear ribosomal RNA genes fo the unicellular eucaryote, Tetrahymena thermophila, the 26S rRNA coding region is interrupted by a 413 base pair intervening sequence (IVS). The IVS is contained within the primary transcript but is subsequently excised from the RNA by splicing. We have recently developed a completely defined, in vitro splicing system. We found that the splicing activity is intrinsic to the IVS portion of the RNA, and that no enzyme or other protein is required. The objective of the proposed research is to further characterize the mechanism of pre-rRNA splicing and the structure of the RNA needed to mediate the reaction. The structure of the junction formed during ligation of the exons (rRNA sequences bordering the IVS) will be determined by RNA fingerprinting, and the kinetics and cofactor requirements of this ligation reaction will be investigated. The binding site of the guanosine cofactor in the RNA will be characterized by kinetic studies with guanosine analogs and by photoaffinity labeling. The secondary structure of the IVS will be determined using chemical and enzymatic probes, computer calculations and phylogenetic comparison of sequences. The structure of the excised IVS RNA will be compared to that of the IVS when it is part of the precursor. Generalized and sitespecific mutagenesis of recombinant plasmid DNA followed by in vitro transcription will be used to produce altered pre-rRNA molecules which will be tested in the in vitro splicing system; this will allow defects in specific steps of the splicing reaction to be correlated with specific changes in the RNA sequence. Finally, the generality of the Tetrahymena pre-rRNA splicing mechanism will be investigated. The Tetrahymena pre-rRNA splicing system is unusually amenable to detailed study. It is hoped that some of the findings will be applicable to the much less tractable problem of human mRNA splicing, defects in which are known to cause diseases such as Beta+-thalassemia.